Potentiation of apoptosis by flavopiridol in mitomycin-C-treated gastric and breast cancer cells.-猫眼课题宝

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1997-09

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Clinical cancer research : an official journal of the American Association for Cancer Research

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Yu‐Xin Hu;Hiroyuki Watanabe;K. Ohtsubo;J. H. Carter;J. Deddens;A. Kaplan;D. Kieback;C. Meyers;Kurt A. Jaeckhe;E. Loyer;M. Steger;A. Killian;Rosa A. Tang;S. Bernstein;J. P. Fay;N. Christiansen;Luis Piñero;D. Shah;M. Stephan;G. Herzig;J. Schellens;L. Webster;Michael Mihlward;J. Verweij;J. Greiner;J. Schlom

Flavopiridol（L 86 -8275）是一种合成黄酮，目前正在进行I期临床试验。它对一系列人类癌细胞系具有活性，并已被证明可抑制广泛的蛋白激酶，包括细胞周期蛋白依赖性激酶和蛋白激酶C（PKC）。以往的研究表明，PKC特异性抑制剂safingol显著增强丝裂霉素C（MMC）诱导胃癌细胞凋亡。因为flavopiridol可以潜在地抑制PKC，我们选择确定flavopiridol在胃癌和乳腺癌细胞中促进MMC诱导的凋亡的程度。在这些研究中，将MKN-74胃癌细胞和MDA-MB-468乳腺癌细胞暴露于无药物、单独的1微克/ml MMC、单独的300 nM flavopiridol或化疗与flavopiridol的组合24小时。还通过首先将细胞暴露于MMC 24小时，然后暴露于flavopiridol 24小时或以相反顺序暴露于相同药物来检查序列特异性。细胞凋亡是通过定量荧光显微镜的染色质凝聚的染料，bisbenzimide trihydrochloride染色的细胞。单独暴露于flavopiridol的MKN-74细胞诱导12 +/- 1%的细胞凋亡，单独暴露于MMC诱导10 +/-1%的细胞凋亡。然而，flavopiridol和MMC的组合将细胞凋亡的诱导增加至55 +/-3%（药物组合与单独的flavopiridol相比P < 0.005）。用PKC激活剂3-佛波醇12-肉豆蔻酸酯13-乙酸酯预处理仅部分逆转这种作用（43 +/- 1%; P < 0.025）。在MDA-MB-468细胞中，单独flavopiridol诱导17 +/- 1%的细胞凋亡，单独MMC诱导10 +/- 1%的细胞凋亡。Flavopiridol和MMC的组合将经历凋亡的MDA-MB-468细胞的百分比增加至58 +/-4%（药物组合与单独的Flavopiridol相比P < 0.005）。MMC和Flavopiridol序贯治疗诱导了63 +/- 2%的MKN-74细胞（与联合用药相比P < 0.05）和76 +/- 2%的MDA-MB-468细胞（与联合用药相比P < 0.025）的凋亡，而Flavopiridol和MMC序贯治疗均未增加两种细胞系的凋亡诱导。如通过末端脱氧核苷酸转移酶标记凋亡细胞中产生的DNA片段的3'末端所确定的，在细胞周期的所有阶段（即，G 0-G1、S和G2-M）。这些结果表明，flavopiridol通过促进肿瘤细胞中药物诱导的凋亡而增强化疗剂MMC的细胞毒性作用。测序研究表明，MMC后flavopiridol或同时治疗优于flavopiridol后MMC的上级。Flavopiridol增强MMC诱导的细胞凋亡可能部分依赖于PKC，并且与细胞周期的一个特定区域无关。

Flavopiridol (L86-8275) is a synthetic flavone currently undergoing Phase I clinical trials. It is active against a series of human cancer cell lines and has been shown to inhibit a broad range of protein kinases, including cyclin-dependent kinases and protein kinase C (PKC). Previous studies have shown that the PKC-specific inhibitor safingol significantly enhances the induction of apoptosis by mitomycin-C (MMC) in gastric cancer cells. Because flavopiridol can potentially inhibit PKC, we elected to determine the extent to which flavopiridol would promote MMC-induced apoptosis in both gastric and breast cancer cells. For these studies, MKN-74 gastric cancer cells and MDA-MB-468 breast cancer cells were exposed to either no drug, 1 microgram/ml MMC alone, 300 nM flavopiridol alone, or a combination of chemotherapy with flavopiridol for 24 h. Sequence specificity was also examined by first exposing cells to MMC for 24 h followed by flavopiridol for 24 h or to the same drugs in the reverse order. Apoptosis was measured by quantitative fluorescence microscopy of nuclear chromatin condensation in cells stained with the dye, bisbenzimide trihydrochloride. Exposure of MKN-74 cells to flavopiridol alone induced apoptosis in 12 +/- 1% of the cells, and exposure to MMC alone induced apoptosis in 10 +/- 1%. However, the combination of flavopiridol and MMC increased the induction of apoptosis to 55 +/- 3% of the cells (P < 0.005 for the drug combination versus flavopiridol alone). Pretreatment with the PKC activator 3-phorbol 12-myristate 13-acetate only partially reversed this effect (43 +/- 1%; P < 0.025). In MDA-MB-468 cells, flavopiridol alone induced apoptosis in 17 +/- 1% of the cells, and MMC alone induced apoptosis in 10 +/- 1% of the cells. The combination of flavopiridol and MMC increased the percentage of MDA-MB-468 cells undergoing apoptosis to 58 +/- 4% (P < 0.005 for the drug combination versus flavopiridol alone). Sequential treatment with MMC followed by flavopiridol induced apoptosis in 63 +/- 2% of the MKN-74 cells (P < 0.05 versus the concomitant drug combination) and in 76 +/- 2% of the MDA-MB-468 cells (P < 0.025 versus the concomitant drug combination), whereas flavopiridol followed by MMC did not increase the induction of apoptosis in either cell line. As determined by the terminal deoxynucleotidyl transferase labeling of the 3' ends of DNA fragments produced in apoptotic cells, the induction of apoptosis with the combination of flavopiridol and MMC occurred to MKN-74 cells in all phases of the cell cycle (i.e., G0-G1, S, and G2-M). These results indicate that flavopiridol potentiates the cytotoxic effect of the chemotherapeutic agent MMC by promoting drug-induced apoptosis in tumor cells. Sequencing studies suggest that MMC followed by flavopiridol or simultaneous treatment is superior to flavopiridol followed by MMC. The enhancement of MMC-induced apoptosis by flavopiridol may be partially PKC dependent and is not associated with one specific region of the cell cycle.

Potentiation of apoptosis by flavopiridol in mitomycin-C-treated gastric and breast cancer cells.

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